NopAA and NopD Signaling Association-Related Gene GmNAC27 Promotes Nodulation in Soybean (Glycine max)

Rhizobia secrete effectors that are essential for the effective establishment of their symbiotic interactions with leguminous host plants. However, the signaling pathways governing rhizobial type III effectors have yet to be sufficiently characterized. In the present study, the type III effectors, NopAA and NopD, which perhaps have signaling pathway crosstalk in the regulation of plant defense responses, have been studied together for the first time during nodulation. Initial qRT-PCR experiments were used to explore the impact of NopAA and NopD on marker genes associated with symbiosis and defense responses. The effects of these effectors on nodulation were then assessed by generating bacteria in which both NopAA and NopD were mutated. RNA-sequencing analyses of soybean roots were further utilized to assess signaling crosstalk between NopAA and NopD. NopAA mutant and NopD mutant were both found to repress GmPR1, GmPR2, and GmPR5 expression in these roots. The two mutants also significantly reduced nodules dry weight and the number of nodules and infection threads, although these changes were not significantly different from those observed following inoculation with double-mutant (HH103ΩNopAA&NopD). NopAA and NopD co-mutant inoculation was primarily found to impact the plant–pathogen interaction pathway. Common differentially expressed genes (DEGs) associated with both NopAA and NopD were enriched in the plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways, and no further changes in these common DEGs were noted in response to inoculation with HH103ΩNopAA&NopD. Glyma.13G279900 (GmNAC27) was ultimately identified as being significantly upregulated in the context of HH103ΩNopAA&NopD inoculation, serving as a positive regulator of nodulation. These results provide new insight into the synergistic impact that specific effectors can have on the establishment of symbiosis and the responses of host plant proteins.


Introduction
Through their symbiotic interactions with leguminous plants, rhizobia can facilitate biological nitrogen fixation that is conducive to more robust plant growth and development, reducing the need to apply chemical fertilizers to crops [1,2].Soybeans are the most widely cultivated legume in the world, serving as a key source of oil and protein for human consumption [3].Large quantities of industrial nitrogen fertilizer are routinely applied to meet the levels of soybean production necessary to meet with current demand [4].However, this fertilizer application poses a serious threat to environmental integrity while also threatening the diversity of soil microbes in affected regions [5][6][7].Research focused on interactions between soybean plants and rhizobia can profile a foundation for the rational application of biological nitrogen fixation, thus providing a more efficient and ecologically sound supply of nitrogen to growing plants [8,9].
The establishment of the symbiotic association between nitrogen-fixing rhizobia and their leguminous hosts is a complex process that necessitates frequent reciprocal signaling between these microbes and the host plants [10].Soybean-derived flavonoids can promote rhizobial synthesis and secretion of nodulation factor (NF) via the upregulation of the NodD gene [11,12].Once activated, NodD binds to the promoter upstream of TtsI, inducing the expression of this gene [13].TtsI is capable of binding to Tts box promoter sequences, thereby inducing the upregulation of type III secretion system (T3SS)-related genes [14].The type III effectors (T3Es) that are secreted by these T3SS systems are closely associated with successful rhizobial colonization.According to the results of the current study, T3Es can be divided into two types: one is structural proteins and the other is nodulation outer proteins (Nops).Indeed, TtsI mutants exhibit markedly suppressed T3E synthesis and release, thus adversely impacting the establishment of symbiotic relationships with host plants.Transcriptomic analyses, extracellular protein characterization, and nodulation testing have all confirmed that the TtsI mutant strain of Sinorhizobium fredii HH103 exhibits impaired T3Es expression and nodulation attributable to this TtsI mutation.In addition to their effects on the establishment of symbiotic relationships, T3Es also govern the induction of plant defense responses, thereby influencing rhizobia colonization efficiency [15][16][17].GmPR1 (pathogenesis-related gene 1) expression was significantly elevated in the roots of Williams 82 soybeans following inoculation with the TtsI mutant strain as compared to the wild type strain [18].Research focused on pathogenic bacteria such as Pseudomonas and Xanthomonas has additionally demonstrated an essential role for T3Es in the incidence of infection [19,20].
To date, a subset of Nop proteins have been identified and subject to biochemical and functional characterization.For example, the T3SS apparatus of Sinorhizobium has been shown to consist of at least three members of this protein family: NopA, NopB, and NopX [21].The Bradyrhizobium USDA110 NopP protein is capable of interacting with the R protein GmNNL1 and promoting the induction of immune responses in hosts [22].The E3 ubiquitin NopM may function by regulating the activity of the MAPK pathway during the establishment of symbiotic relationships [23].The glycoside hydrolase 12 (GH12) family glycosyl hydrolase NopAA can hydrolyze the cell wall components β-glucan and xyloglucan into sugars, thus favoring rhizobia infection [24].It can further influence nodule numbers by shaping the number of infection threads, differentially impacting the nodulation of soybean cultivars with distinct genetic characteristics [24].
The functional importance of T3SS activity in the context of symbiosis is generally not dependent on any specific T3E, instead arising from the redundant, synergistic, or antagonistic interactions of various T3Es [25,26].Deleting a given T3E may thus enhance or inhibit nodulation [9,27], but it may also have no impact on this process.Deleting Bel2-5, ErnA, NopAB, NopC, NopD, NopE, NopF, NopI, NopJ, NopL, NopM, NopP, or InnB can reportedly inhibit nodulation [28][29][30][31], but the impact of specific T3Es on the process of nodule formation is also believed to be host plant-dependent [32][33][34][35].For example, deleting NopT in Ensifer fredii strain NGR234 can interfere with Tephrosia vogelii nodule formation while enhancing such nodulation in Crotalaria juncea [36].Particular T3Es can interact in concert to shape symbiotic nodulation.For example, the double mutation of NopL and NopP can interfere with nodulation in Flemingia congesta to a greater degree than the single mutation of either of these Nop genes in NGR234 [37].Similarly, the double mutation of nopP1 and nopM1 in Bradyrhizobium vignae ORS3257 had a more pronounced impact on nodule formation [30,38].While NopT and NopP reportedly serve as respective promoters and inhibitors of nodulation, the inoculation of soybean plants with the HH103ΩNopT&NopP strain has been shown to result in the formation of fewer nodules than inoculation with the HH103ΩNopT strain [39].RNA-seq analyses have also highlighted a potential role for GmPBS1, which interacts with HH103-derived NopT, in the signaling crosstalk between NopT and NopP [39].No differences in nodule formation were observed when compar-ing HH103ΩNopL&NopT inoculation to HH103ΩNopL or HH103ΩnopP [40].QTL and RNA-seq analyses have also highlighted several NopT and NopL-related genes [40].
Prior RNA-seq and QTL screening experiments have identified a number of NopAAregulated genes in soybean plants, including the defense response-related GmPR1 gene as well as members of the ethylene response factor (ERF) and WRKY transcription factor families [41][42][43].NopD is a positive regulator of nodulation first identified in S. fredii HH103 culture supernatants [44].It harbors a C-terminal structural domain homologous to the ubiquitin-like proteinase Ulp1, and the homologous Xanthomonas protein XopD can interact with small ubiquitin-like modifier (SUMO)-binding proteins to facilitate the removal of SUMO-binding plant proteins, indicating that NopD may play a similar role [45].In Arabidopsis thaliana, XopD can suppress immune response activity [46], whereas NopD reportedly promotes programmed cell death in tobacco [44].The NopD homolog Bradyrhizobium elkanii T3E Bel2-5 can further regulate nodule numbers and the differential expression of a wide range of redox-related genes [28].The ability of NopD, XopD, and Bel2-5 to interact with host plants is closely tied to the ULP1 domain [28,45,47].Much as with NopAA, mutations in Bel2-5 modulate the expression of several soybean genes including ERF1b, ERF98, and WRKY33/75 [28].These results suggest that both NopAA and NopD may be involved in the regulation of plant defense responses during the establishment of symbiosis and may be related in some signaling pathways.
In the present study, the inoculation of soybean plants with both NopAA mutant and NopD mutant strains was found to reduce the expression of the defense-related GmPR1, GmPR2, and GmPR5 following rhizobial infection.While HH103ΩNopAA&NopD inoculation similarly reduced soybean nodulation, it did so to an extent that was not significantly different from that observed for HH103ΩNopAA or HH103ΩNopD.RNA-seq analysis revealed that the expression of a range of plant-pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway-related genes was similarly impacted by NopAA and NopD.The NAC family transcription factor Glyma.13G279900 (GmNAC27) was subsequently identified through WGCNA and qRT-PCR experiments as a candidate gene associated with these two T3Es.Transgene analyses then revealed that GmNAC27 was responsive to the co-associative effects of NopAA and NopD, serving as a positive regulator of nodulation activity.Together, these results provide new insight into the mechanistic basis for legume-rhizobia interactions.

HH103ΩNopAA&D Inhibits Nodule Formation
The ability to successfully evade the host defense response is important for successful colonization of rhizobia [48].Similar to pathogenic bacteria, rhizobia utilize type III effectors to suppress host defenses or activate symbiotic responses.To analyze the host signaling pathways in which the type III effectors NopAA and NopD are mainly involved, in the present study the changes in symbiosis-related genes (GmNIN, GmENOD40, and GmNSP1) and defense-related genes (GmPR1, GmPR2, and GmPR5) expression in SN14 following HH103, HH103ΩNopAA, or HH103ΩNopD inoculation were next assessed at 36 h post-infection [1,49].In these analyses, significantly lower levels of GmPR1, GmPR2, and GmPR5 expression were detected in SN14 plants following NopAA mutant or NopD mutant inoculation as compared to parental HH103 inoculation (Figure 1).The NopAA mutant also induced higher levels of GmNSP1 expression (Figure 1).This suggests that both of these T3Es may play a role in the establishment of symbiosis through their ability to regulate defensive responses.

Figure 1.
The impact of HH103 and mutants' inoculation of nodulation-related gene expression.The relative expression levels of symbiosis-related genes (GmNIN, GmENOD40, and GmNSP1) and defense-related genes (GmPR1, GmPR2, and GmPR5) were identified, and the 2 −ΔΔCt method was used to calculate relative gene expression, with GmUNK1 (Glyma.12g020500)serving as an internal control gene.The calibration samples were SN14 roots inoculated with MgSO4 and used for normalization.Results are means ± SEM from three replicates.Significance was determined by multifactorial analysis of variances (ANOVAs), "**" represent significant differences (p < 0.05), while "ns" indicate no significant differences.
To build on these results, NopD was next mutated in the HH103ΩNopAA strain to yield the HH103ΩNopAA&D mutant strain, with SN14 nodule phenotypes then being evaluated following inoculation with this strain, single mutant strains, or parental HH103.Significant reductions in number of nodules and nodules dry weight were observed following HH103ΩNopAA, HH103ΩNopD, or HH103ΩNopAA&D inoculation as compared to HH103 inoculation (Figure 2A,C).However, no differences in these values were noted when comparing the single-and double-mutant HH103 strains (Figure 2A,C).Toluidine blue staining of nodule cross-sections (NCS) similarly revealed no significant differences in infected cell density within these nodules (Figure 2A).Infection thread phenotypes were additionally evaluated following inoculation with these different HH103 strains expressing the GUS reporter gene.Significant reductions in infection thread numbers were noted following SN14 inoculation with the NopAA mutant, NopD mutant, or NopAA&D mutant strains as compared to HH103 inoculation (Figure 2B,C).This suggests that both NopAA and NopD can suppress nodulation by inhibiting rhizobial infection.Consistently, no significant differences in the impact of NopAA mutant, NopD mutant, or NopAA&D mutant inoculation were observed on infection threads events (Figure 2B,C).This suggests that the NopAA and NopD co-mutant inoculation does not further inhibit nodulation compared to NopAA mutant or NopD mutant, which means that NopAA and NopD might exist as redundant functions in nodulation.The relative expression levels of symbiosis-related genes (GmNIN, GmENOD40, and GmNSP1) and defense-related genes (GmPR1, GmPR2, and GmPR5) were identified, and the 2 −∆∆Ct method was used to calculate relative gene expression, with GmUNK1 (Glyma.12g020500)serving as an internal control gene.The calibration samples were SN14 roots inoculated with MgSO 4 and used for normalization.Results are means ± SEM from three replicates.Significance was determined by multifactorial analysis of variances (ANOVAs), "**" represent significant differences (p < 0.05), while "ns" indicate no significant differences.
To build on these results, NopD was next mutated in the HH103ΩNopAA strain to yield the HH103ΩNopAA&D mutant strain, with SN14 nodule phenotypes then being evaluated following inoculation with this strain, single mutant strains, or parental HH103.Significant reductions in number of nodules and nodules dry weight were observed following HH103ΩNopAA, HH103ΩNopD, or HH103ΩNopAA&D inoculation as compared to HH103 inoculation (Figure 2A,C).However, no differences in these values were noted when comparing the single-and double-mutant HH103 strains (Figure 2A,C).Toluidine blue staining of nodule cross-sections (NCS) similarly revealed no significant differences in infected cell density within these nodules (Figure 2A).Infection thread phenotypes were additionally evaluated following inoculation with these different HH103 strains expressing the GUS reporter gene.Significant reductions in infection thread numbers were noted following SN14 inoculation with the NopAA mutant, NopD mutant, or NopAA&D mutant strains as compared to HH103 inoculation (Figure 2B,C).This suggests that both NopAA and NopD can suppress nodulation by inhibiting rhizobial infection.Consistently, no significant differences in the impact of NopAA mutant, NopD mutant, or NopAA&D mutant inoculation were observed on infection threads events (Figure 2B,C).This suggests that the NopAA and NopD co-mutant inoculation does not further inhibit nodulation compared to NopAA mutant or NopD mutant, which means that NopAA and NopD might exist as redundant functions in nodulation.
This approach revealed that inoculation with the NopAA mutant, NopD mutant, and NopAA&D mutant strains was associated with 1328 (428 upregulated, 900 downregulated), 1243 (523 upregulated, 720 downregulated), and 1839 (820 upregulated, 1019 downregulated) DEGs, respectively (Figures 3C,D and S1).Based on these results, it appears that HH103ΩNopAA and HH103ΩNopD inoculation induces highly overlapping DEG profiles in soybean roots, while HH103ΩNopAA&NopD inoculation induces even higher numbers of DEGs when comparing gene expression profiles to those induced by Mock or HH103 inoculation.

NopAA and NopD Co-Mutation Primarily Impacts the Plant-Pathogen Interaction Pathway
KEGG and GO enrichment analyses were performed on DEGs to determine their signaling pathways and basic functions.Considerable number of DEGs related to NopAA were mainly enriched in phenylpropanoid biosynthesis, MAPK signaling and pentose and glucuronate interconversion signaling pathways (Figure 4A).NopD mainly affects genes enriched in the plant-pathogen interaction, plant hormone signaling transduction, and phenylpropanoid biosynthesis pathways (Figure 4B).Compared with HH103, the inoculation of the HH103ΩNopAA&NopD caused differences in the expression of genes enriched mainly in plant-pathogen interaction, MAPK signaling pathway, and plant To examine the impact of NopAA and NopD on gene expression in soybean roots, DEGs induced by inoculation with the HH103ΩNopAA, HH103ΩNopD, and HH103ΩNopAA&D were identified through a comparison with HH103-inoculated roots.This approach revealed that inoculation with the NopAA mutant, NopD mutant, and NopAA&D mutant strains was associated with 1328 (428 upregulated, 900 downregulated), 1243 (523 upregulated, 720 downregulated), and 1839 (820 upregulated, 1019 downregulated) DEGs, respectively (Figures 3C,D andt S1).Based on these results, it appears that HH103ΩNopAA and HH103ΩNopD inoculation induces highly overlapping DEG profiles in soybean roots, while HH103ΩNopAA&NopD inoculation induces even higher numbers of DEGs when comparing gene expression profiles to those induced by Mock or HH103 inoculation.

NopAA and NopD Co-Mutation Primarily Impacts the Plant-Pathogen Interaction Pathway
KEGG and GO enrichment analyses were performed on DEGs to determine their signaling pathways and basic functions.Considerable number of DEGs related to NopAA were mainly enriched in phenylpropanoid biosynthesis, MAPK signaling and pentose and glucuronate interconversion signaling pathways (Figure 4A).NopD mainly affects genes enriched in the plant-pathogen interaction, plant hormone signaling transduction, and phenylpropanoid biosynthesis pathways (Figure 4B).Compared with HH103, the inoculation of the HH103ΩNopAA&NopD caused differences in the expression of genes enriched mainly in plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction pathways (Figure 4C).And these DEGs clusters were assigned to three broad categories: biological processes, cellular components, and molecular functions; most genes in these categories were enriched in cellular processes, metabolic process, and single-organism processes; cells and cell parts and organelles; and binding, catalytic activity, and transporter activity, respectively (Figure 4A-C).
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 7 of 19 hormone signal transduction pathways (Figure 4C).And these DEGs clusters were assigned to three broad categories: biological processes, cellular components, and molecular functions; most genes in these categories were enriched in cellular processes, metabolic process, and single-organism processes; cells and cell parts and organelles; and binding, catalytic activity, and transporter activity, respectively (Figure 4A-C).(A-C) KEGG and GO enrichment analysis results for genes differentially expressed when comparing HH103ΩNopAA (A), HH103ΩNopD (B), and HH103ΩNopAA&D (C) inoculation to HH103 inoculation.

NopAA and NopD Have Similar Effects on the Plant-Pathogen Interaction Pathway
To better understand the signaling crosstalk between NopAA and NopD, the overlapping DEGs between the HH103ΩNopAA vs. HH103 and HH103ΩNopD vs. HH103 comparisons were next identified.Relative to HH103ΩNopAA and HH103ΩNopD inoculation, HH103ΩNopAA&NopD inoculation did further impact the expression of the 434 common DEGs (121 upregulated, 313 downregulated) (Figures 3A,B and 5A,C).KEGG enrichment analyses of the common upregulated DEGs revealed that they were primarily enriched in the phenylpropanoid biosynthesis and plant-pathogen interaction signaling pathways (Figure 5D), whereas many of the common downregulated DEGs were enriched in the MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction pathways (Figure 5B).Strikingly, several of these common DEGs are also associated with XopD, which is also released by bacterial secretion systems (Figure S2).These data highlight potential crosstalk between NopAA and NopD with respect to their effects on downstream plant-pathogen interaction, MAPK signaling, and plant hormone signal transduction activity in soybean roots.

NopAA and NopD Have Similar Effects on the Plant-Pathogen Interaction Pathway
To better understand the signaling crosstalk between NopAA and NopD, the overlapping DEGs between the HH103ΩNopAA vs. HH103 and HH103ΩNopD vs. HH103 comparisons were next identified.Relative to HH103ΩNopAA and HH103ΩNopD inoculation, HH103ΩNopAA&NopD inoculation did further impact the expression of the 434 common DEGs (121 upregulated, 313 downregulated) (Figures 3A,B and 5A,C).KEGG enrichment analyses of the common upregulated DEGs revealed that they were primarily enriched in the phenylpropanoid biosynthesis and plant-pathogen interaction signaling pathways (Figure 5D), whereas many of the common downregulated DEGs were enriched in the MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction pathways (Figure 5B).Strikingly, several of these common DEGs are also associated with XopD, which is also released by bacterial secretion systems (Figure S2).These data highlight potential crosstalk between NopAA and NopD with respect to their effects on downstream plant-pathogen interaction, MAPK signaling, and plant hormone signal transduction activity in soybean roots.

WGCNA Analyses Reveal Candidate NopAA and NopD-Associated Genes
While NopAA and NopD co-mutant inoculation did not significantly impact the overlapping DEGs associated with these effectors, a number of DEGs were also identified in soybean roots in response to HH103ΩNopAA&D inoculation.To better clarify the genes that were responsive to NopAA and NopD co-mutant inoculation, all RNA-seq data were next utilized to conduct a weighted correlation network analysis (WGCNA).In total, 1428 genes were grouped into nine co-expressed gene clusters based upon their correlational relationships, and these modules were assigned different colors (Figure 6A,B).Correlations among modules were evaluated with a module eigengene adjacency heatmap, which revealed that genes in the red module were specifically expressed in the roots following HH103ΩNopAA&D inoculation (Figure 6A).KEGG enrichment analyses indicated that the genes in the red module were primarily enriched in the plant hormone signal transduction pathway (Figure 6C).In terms of GO term enrichment, these red module genes were primarily enriched in the "metabolic process, cellular processes, and single-organism processes" biological process terms, the "cells and cell parts and organelles" cellular component terms, and the "binding, catalytic activity, and transporter activity" molecular function terms (Figure 6D).Analyses of the FPKM values for the hub genes in this red module were conducted (Figure S3), revealing that these genes were significantly repressed in roots inoculated with HH103 & NopAA (Table S3).

WGCNA Analyses Reveal Candidate NopAA and NopD-Associated Genes
While NopAA and NopD co-mutant inoculation did not significantly impact the overlapping DEGs associated with these effectors, a number of DEGs were also identified in soybean roots in response to HH103ΩNopAA&D inoculation.To better clarify the genes that were responsive to NopAA and NopD co-mutant inoculation, all RNA-seq data were next utilized to conduct a weighted correlation network analysis (WGCNA).In total, 1428 genes were grouped into nine co-expressed gene clusters based upon their correlational relationships, and these modules were assigned different colors (Figure 6A,B).Correlations among modules were evaluated with a module eigengene adjacency heatmap, which revealed that genes in the red module were specifically expressed in the roots following HH103ΩNopAA&D inoculation (Figure 6A).KEGG enrichment analyses indicated that the genes in the red module were primarily enriched in the plant hormone signal transduction pathway (Figure 6C).In terms of GO term enrichment, these red module genes were primarily enriched in the "metabolic process, cellular processes, and single-organism processes" biological process terms, the "cells and cell parts and organelles" cellular component terms, and the "binding, catalytic activity, and transporter activity" molecular function terms (Figure 6D).Analyses of the FPKM values for the hub genes in this red module were conducted (Figure S3), revealing that these genes were significantly repressed in roots inoculated with HH103 & NopAA (Table S3).

qRT-PCR Verification of Candidate Genes
Next, the hub genes identified within the red module were annotated, and relative changes in their expression were assessed in the roots of soybean plants following inoculation with mutant or parental HH103 strains.Of the analyzed genes, Glyma.13G279900 was found to be upregulated to a significantly higher level in roots inoculated with HH103ΩNopAA&D as compared to roots inoculated with HH103 (Figure 7), whereas its expression was not significantly impacted by inoculation with HH103ΩNopAA as compared to HH103, although it was significantly upregulated in HH103ΩNopD-inoculated roots.No other analyzed genes exhibited significant upregulation in response to HH103ΩNopAA&D inoculation.

qRT-PCR Verification of Candidate Genes
Next, the hub genes identified within the red module were annotated, and relative changes in their expression were assessed in the roots of soybean plants following inoculation with mutant or parental HH103 strains.Of the analyzed genes, Glyma.13G279900 was found to be upregulated to a significantly higher level in roots inoculated with HH103ΩNopAA&D as compared to roots inoculated with HH103 (Figure 7), whereas its expression was not significantly impacted by inoculation with HH103ΩNopAA as compared to HH103, although it was significantly upregulated in HH103ΩNopD-inoculated roots.No other analyzed genes exhibited significant upregulation in response to HH103ΩNopAA&D inoculation.
Figure 7. qRT-PCR-based validation of NopAA&D mutant-induced hub genes expression in soybean roots.GmUNK1 (Glyma.12g020500)served as an internal control gene.The calibration samples were SN14 roots inoculated with MgSO4 and used for normalization.Data are presented as means with standard deviations.Significance was determined by multifactorial analysis of variances (ANOVAs), different letters represent significant differences (p < 0.05), while the same letters indicate no significant differences.

Glyma.13G279900 Is a NAC Family Transcription Factor Located on the Cell Nucleus
Glyma.13G279900 encodes the NAC family transcription factor GmNAC27. Phylogenetic analyses revealed a close evolutionary relationship between GmNAC27 and similar genes in A. thaliana (Figure S4).A subcellular localization analysis of GmNAC27 conducted by infiltrating Nicotiana benthamiana leaves with Agrobacterium tumefaciens EHA105 harboring the CaMV35S: GmNAC27:GFP fusion vector revealed that the GmNAC27-GFP fusion protein is primarily localized to the nucleus in infected cells (Figure S5).

Analyses of the Impact of GmNAC27 RNA-Interference and Overexpression on Soybean Nodulation
To establish the impact of GmNAC27 on symbiotic nodulation in soybean plants, this gene was next silenced or overexpressed via soybean transgenic hairy root transformation using Agrobacterium rhizogenes strain K599 carrying the pB7GWWIWG2(II)-DsRed-GmNAC27 or pSoy10-GmNAC27-GFP plasmid constructs, respectively.Successful GmNAC27 overexpression or RNAi was confirmed via qRT-PCR (Figure S6).Hairy roots overexpressing GmNAC27 exhibited significantly higher numbers of nodules and nodule dry weight relative to control plants following HH103, NopAA, or NopD single mutant inoculation, with nodulation remaining more robust following parental HH103 inoculation as compared to inoculation with either mutant strain (Figure 8).However, no significant differences in nodule number or dry weight were noted when comparing GmNAC27-OE hairy roots inoculated with HH103ΩNopAA&D and the corresponding EV1 empty vector control (Figure 8).Significant reductions in nodule number and dry weight were observed for hairy roots in which GmNAC27 had been silenced following HH103, HH103ΩNopAA, HH103ΩNopD, or HH103ΩNopAA&D inoculation as compared to corresponding EV2 control plants (Figure 8).Analyses of symbiosis and defense marker gene

Glyma.13G279900 Is a NAC Family Transcription Factor Located on the Cell Nucleus
Glyma.13G279900 encodes the NAC family transcription factor GmNAC27. Phylogenetic analyses revealed a close evolutionary relationship between GmNAC27 and similar genes in A. thaliana (Figure S4).A subcellular localization analysis of GmNAC27 conducted by infiltrating Nicotiana benthamiana leaves with Agrobacterium tumefaciens EHA105 harboring the CaMV35S: GmNAC27:GFP fusion vector revealed that the GmNAC27-GFP fusion protein is primarily localized to the nucleus in infected cells (Figure S5).

Analyses of the Impact of GmNAC27 RNA-Interference and Overexpression on Soybean Nodulation
To establish the impact of GmNAC27 on symbiotic nodulation in soybean plants, this gene was next silenced or overexpressed via soybean transgenic hairy root transformation using Agrobacterium rhizogenes strain K599 carrying the pB7GWWIWG2(II)-DsRed-GmNAC27 or pSoy10-GmNAC27-GFP plasmid constructs, respectively.Successful GmNAC27 overexpression or RNAi was confirmed via qRT-PCR (Figure S6).Hairy roots overexpressing GmNAC27 exhibited significantly higher numbers of nodules and nodule dry weight relative to control plants following HH103, NopAA, or NopD single mutant inoculation, with nodulation remaining more robust following parental HH103 inoculation as compared to inoculation with either mutant strain (Figure 8).However, no significant differences in nodule number or dry weight were noted when comparing GmNAC27-OE hairy roots inoculated with HH103ΩNopAA&D and the corresponding EV1 empty vector control (Figure 8).Significant reductions in nodule number and dry weight were observed for hairy roots in which GmNAC27 had been silenced following HH103, HH103ΩNopAA, HH103ΩNopD, or HH103ΩNopAA&D inoculation as compared to corresponding EV2 control plants (Figure 8).Analyses of symbiosis and defense marker gene expression in transgenic roots were also assessed, revealing that GmNAC27 had no effect on symbiosis marker genes or GmPR2, whereas it was able to positively regulate GmPR1 and GmPR5 expression (Figure S7).These results suggest that GmNAC27 may play a role in the signaling crosstalk between the T3Es NopAA and NopD through its ability to regulate defense responses and to serve as a positive regulator of nodulation.expression in transgenic roots were also assessed, revealing that GmNAC27 had no effect on symbiosis marker genes or GmPR2, whereas it was able to positively regulate GmPR1 and GmPR5 expression (Figure S7).These results suggest that GmNAC27 may play a role in the signaling crosstalk between the T3Es NopAA and NopD through its ability to regulate defense responses and to serve as a positive regulator of nodulation.

Discussion
The initial analyses conducted herein confirmed that NopAA and NopD similarly impacted the expression of the GmPR1, GmPR2, and GmPR5 marker genes during rhizobia infection, while NopAA and NopD co-mutant inoculation had no significant effects on nodule formation beyond those observed in the context of inoculation with mutant strains for either of these genes individually.Relative to HH103 inoculation, the inoculation of

Discussion
The initial analyses conducted herein confirmed that NopAA and NopD similarly impacted the expression of the GmPR1, GmPR2, and GmPR5 marker genes during rhizobia infection, while NopAA and NopD co-mutant inoculation had no significant effects on nodule formation beyond those observed in the context of inoculation with mutant strains for either of these genes individually.Relative to HH103 inoculation, the inoculation of soybean plants with the NopAA mutant and NopD mutant strains resulted in the differential expression of a large number of genes associated with the plant-pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways.GmNAC27 was ultimately identified as a soybean protein that was differentially expressed in response to the concurrent deletion of NopAA and NopD.Together, these results offer a new foundation for research focused on clarifying the mechanistic crosstalk between signaling pathways and associated regulatory mechanisms associated with the function of NopAA and NopD in the establishment of host-rhizobia symbiosis.
First characterized through studies of pathogenic bacteria, T3SS-mediated T3E secretion can enable these pathogens to evade plant immune responses [50].Plants have evolved the ETI response to T3E exposure, which typically manifests in the form of a robust hypersensitivity reaction resulting in cell death [51].Rhizobia-derived T3Es often regulate immune functionality in host plants in addition to supporting the establishment of symbiosis [52].In this study, the relative levels of nodulation maker genes (GmNIN [53], GmENOD40 [54], GmNSP1 [55]) and defense-related genes (GmPR1 [8], GmPR2, GmPR5) were assessed to better clarify the effects of NopAA and NopD on host genes expression.Inoculation with both the NopAA mutant and the NopD mutant strains resulted in significant reductions in GmPR1, GmPR2, and GmPR5 expression, with this suppression being most pronounced for the NopAA mutant.This stronger impact of NopAA on host defense gene expression may be related to its ability to hydrolyze cell walls, thereby inducing more robust immune response activity [24].The fact that NopD regulates defensive responses via programmed cell death pathways may also contribute to this observation [44].Together, these findings suggest that NopAA and NopD primarily play roles in the modulation of defensive responses in soybean roots, rather than directly influencing nodulation-related symbiotic signals.
The effects of T3SS activity in the context of symbiotic interactions are not dependent on the effects of a single T3E, instead arising as a result of redundant, synergistic, or antagonistic effects among multiple T3Es [26].With the exception of the complete inhibition of Rj2-soybean nodulation in response to NopP in USDA122, there have not been any reports of any one T3E completely determining whether or not nodulation can occur [56].In previous studies, our team has utilized HH103 T3E gene insertion mutants and a variety of soybean genetic resources to assess nodulation dynamics, revealing soybean varietyspecific effects of these T3Es [21,24,44].Both NopAA and NopD were previously found to be likely contributors to the nodulation process.To better understand how NopAA and NopD impact nodulation, the HH103ΩNopAA&NopD strain was generated in this present study.HH103ΩNopAA&NopD inoculation was associated with a reduction in the numbers of infection threads and nodules, although these effects did not differ significantly from the phenotypes observed following NopAA mutant or NopD mutant inoculation.This suggests that NopAA and NopD serve as redundant regulators of nodulation or that crosstalk between the signaling pathways downstream of these effectors shapes nodule formation.Both NopAA and NopD ultimately impact nodulation via their effects on rhizobial infection, and while both of these T3Es can promote nodulation, neither appears to be required for nodulation.
T3Es can exert their functions when secreted into plants, whereupon host proteins can respond to or interact with these effectors.Both genetic and RNA-seq analyses have been utilized to identify the downstream response pathways and interacting proteins associated with these T3Es [21,24,57,58].Here, RNA-seq analyses revealed that NopAA can induce substantial numbers of DEGs enriched in the MAPK pathway, in line with prior data related to rhizobia infection.NopAA does not induce necrosis in tobacco leaves [24], and it impacts GmCDPK28 and GmWRKY33 expression [42], suggesting that it may play a role in shaping plant defense responses through the regulation of PTI.NopAA was also found to influence many phenylpropanoid biosynthesis-related genes.Phenylpropanoid metabolic pathway-derived lignin is a primary component of cell walls in plants, and NopAA-mediated cellulose hydrolysis can cause cell wall damage and impact lignin metabolism [24].The C-terminal region of NopD from S. fredii HH103 harbors a critical functional domain with a sequence similar to that of Bel2-5 and XopD [45].Bel2-5 functions as a promoter of nodulation through its ability to regulate cytokinin biosynthesis and ethylene biosynthesis [28], whereas XopD plays a role in shaping host salicylic acid, gibberellic acid, abscisic acid, and ethylene signaling pathway activity [46].NopD was also found to impact large numbers of genes enriched in the plant hormone signal transduction signaling pathway.In tobacco, NopD can trigger ETI-like programmed cell death [44], potentially consistent with its role in the plant-pathogen interaction signaling pathway.Much like the HH103ΩNopAA strain, inoculation with the HH103ΩNopD strain was also herein found to result in the differential expression of a large number of phenylpropanoid biosynthesisrelated genes.Altered cell wall modification-and xyloglucan metabolism-related gene expression has also been observed in response to bel2-5 deletion mutants [28].When utilizing HH103 inoculation as the comparator, many overlapping DEGs were identified between NopAA-mutant-inoculated and NopD-mutant-inoculated roots that were related to phenylpropanoid biosynthesis.While NopAA mutant inoculation did not result in the differential expression of many plant hormone signal transduction-related DEGs, a large number of hormone signal transduction-related DEGs were identified when assessing the overlapping DEGs between NopAA-mutant-inoculated and NopD-mutant-inoculated roots.These results are consistent with potential functional redundancy between NopD and NopAA with respect to their effects on hormone signal transduction.The greatest proportion of overlapping DEGs between NopAA-mutant-inoculated and NopD-mutant-inoculated roots were enriched in the plant-pathogen interaction pathway, and a large number of overlapping DEGs were noted with respect to MAPK pathway enrichment.Many of these overlapping DEGs were also enriched in XopD-associated pathways, suggesting that there is a degree of gene network redundancy or synergistic regulatory efficacy between NopAA and NopD with respect to the regulation of interactions between soybean plants and HH103 rhizobia.HH103ΩNopAA&NopD inoculation did not further modulate the expression of these overlapping DEGs, nor did it further inhibit nodulation.This may partially explain why HH103&NopAA&NopD inoculation did not further inhibit nodulation.
Inoculation with HH103ΩNopAA&NopD was found to specifically elicit the greatest number of DEGs as compared to HH103, suggesting that when NopAA and NopD are both mutated, a greater number of soybean host genes are engaged to respond as a means of maintaining symbiotic nodulation.Using a WGCNA approach to identify soybean genes that were specifically responsive to HH103ΩNopAA&NopD inoculation, GmNAC27 was identified as ultimately confirmed to be significantly induced by this mutant strain in qRT-PCR analyses.GmNAC27 is a root-specific member of the plant-specific NAC transcription factor family that shares a high degree of homology with the A. thaliana AtNAC072 and AtNAC3 proteins.AtNAC072, also referred to as RESPONSIVE TO DESICCATION 26 (RD26), can enhance the ABA-dependent drought tolerance of plants.AtNAC072 is reportedly upregulated in response to PGN, LPS, and flg22 treatment, suggesting that ANAC072 can respond to MAMP signaling [59].AtNAC3 serves as a promoter of phytohormone synthesis that can bolster anti-pathogen defenses while repressing growth [60].Infection with pathogens can reduce lncRNA SABC1 accumulation, thus alleviating its ability to repress AtNAC3 expression such that this gene is upregulated.When expressed, AtNAC3 can bind the promoter region upstream of ICS1, thereby promoting the upregulation of this key salicylic acid biosynthesis-related gene.GmSIN is a closely related gene cloned from the Shengdou 9 soybean cultivar that reportedly enhances soybean salt tolerance [61].Here, GmNAC27 overexpression in HH103-inoculated hairy roots was found to promote nodulation, suggesting that GmNAC27 serves as a positive regulator of nodule formation.The nodulation ability of GmNAC27-overexpressing hairy roots inoculated with HH103ΩNopAA&D did not differ significantly from that of control plants, while nodulation was significantly inhibited in GmNAC27-RNAi hairy roots inoculated with HH103ΩNopAA&D.This suggests that the absence of both NopAA and NopD, which positively regulate nodulation, may result in the upregulation of GmNAC27 to facilitate the establishment of symbiosis.With respect to the mechanisms responsible for the up-regulation of GmNAC27, it may be that host plants respond by overexpressing GmNAC27 to maintain some level of nodulation, or it may be that NopAA and NopD synergistically repress GmNAC27 such that it is upregulated when both of these effectors are absent.GmNAC27 was found to primarily impact the expression of the defense marker genes GmPR1 and GmPR5, suggesting that it may shape defense response activity in the context of the establishment of symbiosis.However, further experiments will be essential to test these hypotheses.The construction of GmNAC27 transgenic soybean plants will enable experiments aimed at directly confirming the functional role of GmNAC27 in the context of symbiosis, and efforts to screen for relevant genetic variations in different soybean populations may enable the breeding of soybean varieties with superior nitrogen-fixing efficiency.

Strains, Vectors and Primers
Sinorhizobium fredii HH103 (hereafter referred to as HH103) and mutants thereof were used to conduct the present study, as was Escherichia coli DH5α.HH103 and mutant strains were cultured with TY medium containing appropriate antibiotics at 28 • C, while E. coli were cultured in LB medium containing appropriate antibiotics at 37 • C. All antibiotics were used at a concentration of 50 µg/mL.Plasmids and primers used to conduct the present study are presented in Tables S1 and S2.

HH103ΩNopAA&NopD Mutant Strain Construction
Triparental hybridization was employed for HH103ΩNopAA&D mutant construction by first constructing the HH103ΩNopAA mutant [24], followed by the NopD mutation to yield HH103ΩNopAA&D.

Infection Event Analyses
SN14 plants were inoculated with HH103 and mutant strains thereof encoding the GUS reporter gene.At 36 h post-inoculation, roots were harvested, and GUS staining was performed by soaking in 1 mg ml −1 X-Gluc solution containing 100 mM potassium phosphate buffer (pH 7.0), 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, and 10 mM EDTA at 37 • C for 12 h.Then, 70% alcohol was used to decolorize stained roots, after which a total of 10 1 cm long lateral roots were collected from each plat for confocal imaging (Zeiss LSM700, Oberkochen, Germany).Three biological replicates, each consisting of 20 plants, were used for these analyses.

Nodulation Test
Cl 2 was used to sterilize the surfaces of SN14 seeds for 12 h, after which they were sown in autoclaved vermiculite.Seedlings were then cultivated in a greenhouse (light/dark: 16 h/8 h, 25 • C) and routinely irrigated with F nutrient solution.HH103, NopAA mutant, NopD mutant, and NopAA&D mutant strains were cultured in liquid TY medium until reaching an OD 600 of 0.6-0.8, at which time 10 mM MgSO 4 solution was used to wash away the culture medium and to adjust the OD 600 to 0.2.
Soybean roots were inoculated with rhizobia during the Vc phase, and nodule numbers and dry weight were assessed on day 28 post-inoculation.ANOVAs were used to test for significant differences among groups.Three biological replicates each consisting of 20 plants were analyzed for these experiments.
Nodule cross-sections were analyzed by embedding mature nodules (28 days postinoculation) in paraffin.These nodules were then deparaffinized, stained using toluidine blue, and imaged under a light microscope (Olympus SZX16, Tokyo, Japan).

qRT-PCR
Following inoculation with appropriate rhizobia, soybean roots were harvested and TranZol Plant (Transgene Co., Beijing, China) was used to extract total RNA from these samples, followed by DNase I treatment to remove genomic DNA.cDNA was then pre-pared with the TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgene Co.), after which the PerfectStart TM Green One-Step qPCR SuperMix and appropriate primers were utilized for qRT-PCR analyses.The relative expression levels of symbiosis-related genes (GmNIN, GmENOD40, and GmNSP1), defense-related genes (GmPR1, GmPR2, and GmPR5) and candidate genes selected from RNA-seq were identified, and genes expression was normalized to the GmUNK1 (Glyma.12g020500)reference gene [24].

RNA-Seq
The purity, integrity, and concentration of isolated RNA samples were assessed with NanoDrop, Agilent 2100 (Santa Clara, CA, USA)), and other appropriate instruments, after which cDNA library preparation was performed and Q-PCR was used for the quantification of library concentrations (effective concentration > 2 nM) to ensure library quality.Different libraries were then pooled based on the target downstream data volume followed by sequencing with an Illumina system (San Diego, CA, USA).All sequencing analyses were conducted by Biomarker (http://www.biomarker.com.cn/(accessed on 15 March 2022)).

Subcellular Localization Analyses
Subcellular localization analyses were performed using 4-week-old Nicotiana benthamiana plants.Electroporation was used to transform A. tumefaciens EHA105 with the CaMV35S-GmNAC27-GFP plasmid, after which the A. tumefaciens culture was adjusted to an OD 600 of 0.2 using infiltration buffer (10 mM MgCl 2 , 10 mM MES-KOH pH 5.6, 150 µM acetosyringone) and injected into the top leaves of tobacco plants.After 48 h, a Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) was utilized to assess RFP and GFP fluorescence [21].

Soybean Hairy Root Transformation
Soybean hairy root transformation was performed using A. rhizogenes strain K599 containing pSoy10-GmARP-GFP, pSoy10-GFP, pB7GWIWG2-GmARP-DsRed, and pB7GWIWG2-DsRed [62].Transgenic root selection was performed based on qRT-PCR results and the use of a portable fluorescent protein excitation light source (LUYOR), with positive hairy roots then being inoculated using HH103, NopAA mutant, NopD mutant, NopAA&D mutant, or MgSO 4 .Nodulation testing was performed at 28 days post-inoculation.Three independent experiments were utilized to assess nodulation phenotypes, with 20 biological replicates per experiment.

Conclusions
In summary, the present results revealed that NopAA and NopD have similar effects on GmPR1, GmPR2, and GmPR5 expression in soybean.Nodule formation was influenced by the mutation of both NopAA and NopD as a consequence of changes in the incidence of rhizobial infection events.Nodulation tests did not reveal any significant differences in SN14 nodulation when comparing the HH103ΩNopAA&D, HH103ΩNopAA, and HH103ΩNopD strains.RNA-seq analyses further suggested the existence of potential signaling relationships between NopAA and NopD, while WGCNA and transgenic analyses revealed that soybean plants respond to the absence of both of these effectors by upregulating GmNAC27.Together, these results provide a foundation for research focused on T3Es signaling networks and offer support for efforts to breed high-yield soybean varieties with superior nitrogen fixation efficiency.

Figure 1 .
Figure 1.The impact of HH103 and mutants' inoculation of nodulation-related gene expression.The relative expression levels of symbiosis-related genes (GmNIN, GmENOD40, and GmNSP1) and defense-related genes (GmPR1, GmPR2, and GmPR5) were identified, and the 2 −∆∆Ct method was used to calculate relative gene expression, with GmUNK1 (Glyma.12g020500)serving as an internal control gene.The calibration samples were SN14 roots inoculated with MgSO 4 and used for normalization.Results are means ± SEM from three replicates.Significance was determined by multifactorial analysis of variances (ANOVAs), "**" represent significant differences (p < 0.05), while "ns" indicate no significant differences.

Figure 3 .
Figure 3. Venn diagrams representing the numbers of DEGs identified in SN14 roots.(A) Downregulated genes with the indicated rhizobial strains as compared to mock inoculation.(B) Upregulated genes with the indicated rhizobial strains as compared to mock inoculation.(C) Downregulated genes with different HH103 mutant strains relative to parental HH103 inoculation.(D) Upregulated genes with different HH103 mutant strains relative to parental HH103 inoculation.

Figure 3 .
Figure 3. Venn diagrams representing the numbers of DEGs identified in SN14 roots.(A) Downregulated genes with the indicated rhizobial strains as compared to mock inoculation.(B) Upregulated genes with the indicated rhizobial strains as compared to mock inoculation.(C) Downregulated genes with different HH103 mutant strains relative to parental HH103 inoculation.(D) Upregulated genes with different HH103 mutant strains relative to parental HH103 inoculation.

Figure 4 .
Figure 4. Functional analyses of genes differentially regulated by different mutant HH103 strains.(A-C) KEGG and GO enrichment analysis results for genes differentially expressed when comparing HH103ΩNopAA (A), HH103ΩNopD (B), and HH103ΩNopAA&D (C) inoculation to HH103 inoculation.

Figure 4 .
Figure 4. Functional analyses of genes differentially regulated by different mutant HH103 strains.(A-C) KEGG and GO enrichment analysis results for genes differentially expressed when comparing HH103ΩNopAA (A), HH103ΩNopD (B), and HH103ΩNopAA&D (C) inoculation to HH103 inoculation.

Figure 6 .
Figure 6.Transcriptomic WCGNA Analyses.(A) Component analysis of the module corresponding to included genes.(B) Gene co-expression network heatmap.(C,D) KEGG (C) and GO (D) enrichment analyses of genes included in red modules.

Figure 6 .
Figure 6.Transcriptomic WCGNA Analyses.(A) Component analysis of the module corresponding to included genes.(B) Gene co-expression network heatmap.(C,D) KEGG (C) and GO (D) enrichment analyses of genes included in red modules.

Figure 7 .
Figure 7. qRT-PCR-based validation of NopAA&D mutant-induced hub genes expression in soybean roots.GmUNK1 (Glyma.12g020500)served as an internal control gene.The calibration samples were SN14 roots inoculated with MgSO 4 and used for normalization.Data are presented as means with standard deviations.Significance was determined by multifactorial analysis of variances (ANOVAs), different letters represent significant differences (p < 0.05), while the same letters indicate no significant differences.

Figure 8 .
Figure 8. Nodule associated with the overexpression or RNAi of GmNAC27.(A) Nodule phenotypes for hairy roots transformed with EV1, OE, EV2, and RNAi constructs following HH103, NopAA mutant, NopD mutant, or NopAA&D mutant inoculation.EV1, Empty vector for GmNAC27 overexpression; OE, GmNAC27 overexpression vector under the control of CaMV35S; EV2, Empty vector for RNAi; RNAi, GmNAC27 silencing.Scale bars: 1 cm (roots), 2 mm (nodules).(B) Quantification of nodules numbers and dry weight.Data are presented as the averages of three biological replicates (n = 20 plants/replicate).Significance was determined by multifactorial analysis of variances (ANOVAs), different letters represent significant differences (p < 0.05), while the same letters indicate no significant differences.

Figure 8 .
Figure 8. Nodule phenotypes associated with the overexpression or RNAi of GmNAC27.(A) Nodule phenotypes for hairy roots transformed with EV1, OE, EV2, and RNAi constructs following HH103, NopAA mutant, NopD mutant, or NopAA&D mutant inoculation.EV1, Empty vector for GmNAC27 overexpression; OE, GmNAC27 overexpression vector under the control of CaMV35S; EV2, Empty vector for RNAi; RNAi, GmNAC27 silencing.Scale bars: 1 cm (roots), 2 mm (nodules).(B) Quantification of nodules numbers and dry weight.Data are presented as the averages of three biological replicates (n = 20 plants/replicate).Significance was determined by multifactorial analysis of variances (ANOVAs), different letters represent significant differences (p < 0.05), while the same letters indicate no significant differences.

Author Contributions:
Conceptualization, C.L. and Q.C.; methodology, Y.W., J.W. and C.L.; investigation, Y.W., X.J., Y.L. and S.M.; data curation, Y.W., S.M., X.J. and C.M.; writing-original draft preparation, Y.W., X.J. and D.X.; writing-review and editing, C.L. and Q.C.; visualization, C.L. and Q.C.; supervision, C.L. and Q.C.; funding acquisition, C.L. and Q.C.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the National Natural Science Foundation of China (Grant numbers: 32072014 and 32272072).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.